Abstract. Several bacterial infections in humans are polymicrobial with Staphylococcus aureus and Pseudomonas frequently co-cultured from the same infection site. Two populations where S. aureus and P. aeruginosa are frequently found together are diabetic skin and soft tissue infections and in the lungs of patients with cystic fibrosis (CF). A common comorbidity associated with CF is CF-related diabetes (CFRD) that is associated with accelerated rates of pulmonary decline and earlier mortality compared to non-diabetic CF patients. Initially the dominant pathogen in CF lungs is S. aureus. However, S. auerus is eventually replaced by P. aeruginosa as the dominant pathogen. Conversely, in patients with CFRD, S. aureus re-emerges in the presence P. aeruginosa where both are responsible for a significant amount of pulmonary infections. Because of the absence of an in vivo model, many labs study interactions between S. aureus and P. aeruginosa in vitro with the goal of defining how they might interact with each other in CF airways. However, these studies have several limitations in that they are outside the context of an immune response and are performed in conditions that do not accurately reflect the dynamic infection microenvironment. Moreover, in vitro studies cannot adequately replicate diabetic infections. Here we describe a novel murine co-infection model that allows us to study the interactions between S. aureus and P. aeruginosa in a dynamic host environment with an intact immune system in the context of normal and diabetic infections. P. aeruginosa does not survive in a mono- infection in our model. However, during co-infection we observed the ability of P. aeruginosa to grow in the presence of S. aureus. Additionally, we observe the ability of P. aeruginosa to kill S. aureus during co-infection in normal mice. Conversely, in diabetic mice, we observed increased growth of both S. aureus and P. aeruginosa compared to normal mice. We also observed increased virulence potential of both species in diabetic co-infection as both invade surrounding tissues and disseminate to peripheral organs. Aim 1 of the is proposal seeks to identify how S. aureus ?terraforms? the co-infection microenvironment to allow for P. aeruginosa growth as well as determine the mechanisms employed by P. aeruginosa to kill S. aureus. We hypothesize that metabolites produced by S. aureus are used by P. aeruginosa as carbon sources for growth during co-infection, and that P. aeruginosa subsequently produces toxic products that kill S. aureus. In aim 2 of this proposal we seek to identify the mechanisms that allow S. aureus to become immune to killing by P. aeruginosa as well as the mechanisms that allow both species to become more virulent in diabetic infections. We hypothesize that excess glucose in the infection environment allows S. aureus to shift its metabolism to resist the toxic products produced by P. aeruginosa. We additionally propose that glucose promotes the expression of virulence factors in both species resulting in enhanced virulence. In total, we seek to understand and define complex microbial interactions in normal and diabetic environments that cannot be tested in conventional models.